The Na+/glucose cotransporter (SGLT1) is the archetypical cotransporter that uses the energy stored in Na+ electrochemical potential gradients across membranes to drive nutrients into cells. SGLT1 is expressed mainly in the brush border membrane of the small intestine where it is responsible for the absorption of 180g of dietary glucose and galactose per day. The coupling of sugar, salt and water transport by SGLT1 provides the rational for the Oral Rehydration Therapy used to treat infectious diarrhea. Mutations in the gene coding for SGLT1 cause Glucose-Galactose-Malabsorption. The goal of this project is to determine how SGLT1 couples sodium, sugar and water transport. The present aims are to 1) use spectroscopic methods such as ATR-FTIR, CD and fluorescence to monitor and determine the extent of ligand induced conformational changes in the protein, 2) identify the substrate- binding sites using photo-affinity labeling and mass spectrometry, and 3) initiate long term studies to resolve the structure of the protein using freeze-fracture electron microscopy to screen for 2D and 3D crystals. These experiments will be carried out on human SGLT1 and related transporters over expressed in E. coli, purified and reconstituted into liposomes. The results will provide a direct test of the alternating access model for sodium/sugar cotransport.